View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Semmelweis Repository

ORIGINAL RESEARCH published: 14 June 2019 doi: 10.3389/fpsyt.2019.00394

Childhood Adversity Moderates the Effects of HTR2A Epigenetic Regulatory Polymorphisms on Rumination

Nora Eszlari 1,2*, Peter Petschner 1,3, Xenia Gonda 2,3,4, Daniel Baksa 1,5, Rebecca Elliott 6,7, Ian Muir Anderson 6,7, John Francis William Deakin 6,7,8, Gyorgy Bagdy 1,2,3 and Gabriella Juhasz 1,5,6

1 Department of Pharmacodynamics, Faculty of Pharmacy, Semmelweis University, Budapest, Hungary, 2 NAP-2-SE New Antidepressant Target Research Group, Hungarian Brain Research Program, Semmelweis University, Budapest, Hungary, 3 MTA-SE Neuropsychopharmacology and Neurochemistry Research Group, Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary, 4 Department of Psychiatry and Psychotherapy, Kutvolgyi Clinical Centre, Semmelweis University, Budapest, Hungary, 5 SE-NAP 2 Genetic Brain Imaging Migraine Research Group, Hungarian Brain Research Edited by: Program, Semmelweis University, Budapest, Hungary, 6 Division of Neuroscience and Experimental Psychology, Faculty Divya Mehta, of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom, 7 Manchester Academic Health Queensland University of Technology, Sciences Centre, Manchester, United Kingdom, 8 Greater Manchester Mental Health NHS Foundation Trust, Manchester, Australia United Kingdom Reviewed by: Ludwig Stenz, Université de Genève, The serotonin system has been suggested to moderate the association between childhood Switzerland maltreatment and rumination, with the latter in its turn reported to be a mediator in the Gabriel R. Fries, depressogenic effect of childhood maltreatment. Therefore, we investigated whether the University of Texas Health Science Center at Houston, associations of two epigenetic regulatory polymorphisms in the HTR2A serotonin receptor United States gene with Ruminative Responses Scale rumination and its two subtypes, brooding and *Correspondence: reflection, are moderated by childhood adversity (derived from the Childhood Trauma Nora Eszlari [email protected] Questionnaire) among 1,501 European white adults. We tested post hoc whether the univ.hu significant associations are due to depression. We also tested the replicability of the significant results within the two subsamples of Budapest and Manchester. We revealed Specialty section: two significant models: both the association of methylation site rs6311 with rumination This article was submitted to Behavioral and Psychiatric Genetics, and that of miRNA binding site rs3125 (supposed to bind miR-1270, miR-1304, miR-202, a section of the journal miR-539 and miR-620) with brooding were a function of childhood adversity, and both Frontiers in Psychiatry interaction findings were significantly present both in the never-depressed and in the ever- Received: 04 March 2019 Accepted: 17 May 2019 depressed group. Moreover, the association of rs3125 with brooding could be replicated Published: 14 June 2019 across the separate subsamples, and remained significant even when controlling for lifetime Citation: depression and the Brief Symptom Inventory depression score. These findings indicate Eszlari N, Petschner P, Gonda X, the crucial importance of involving stress factors when considering endophenotypes and Baksa D, Elliott R, Anderson IM, Deakin JFW, Bagdy G and Juhasz G suggest that brooding is a more promising endophenotype than a broader measure of (2019) Childhood Adversity rumination. Transdiagnostic relevance of the brooding endophenotype and the potential Moderates the Effects of HTR2A Epigenetic Regulatory of targeting epigenetic regulatory polymorphisms of HTR2A in primary and secondary Polymorphisms on Rumination. prevention of depression and possibly of other disorders are also discussed. Front. Psychiatry 10:394. doi: 10.3389/fpsyt.2019.00394 Keywords: childhood stress, rumination, brooding, serotonin system, perseverative thought

Frontiers in Psychiatry | www.frontiersin.org 1 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

INTRODUCTION (20, 21), and WCST performance correlates negatively with rumination (1). Rs6313 is in high linkage disequilibrium (LD) Ruminative response style (rumination) is a passive and repetitive with another methylable SNP in the promoter region of the gene, way of responding to distress and depressed mood, and it predicts rs6311 (-1438 A/G) (19, 22–24). Rs6313 T allele corresponds to future depression (1). It has two subtypes: the more maladaptive rs6311 T allele (this latter one is measured on the positive strand brooding and the less maladaptive reflection 2( ). Brooding of the DNA double helix, https://genome.ucsc.edu/) (25). Rs6311 means passive comparisons with unachieved standards, whereas also entails controversial phenotypic associations. Its T allele reflection denotes purposeful problem-solving strategies 2( ). is related to an increased promoter function (15, 26–28) and It has been suggested that the depressogenic effect of childhood mood disorders in one study (26) but unrelated to either major maltreatment is partly mediated by rumination (3). However, both depression or bipolar depression according to meta-analyses (29, retrospective studies (4, 5) and also a longitudinal one (6) indicates 30); nevertheless, its CC genotype (defined also on the positive that this mediatory effect is carried by the brooding but not the strand) is the one that confers a risk for higher neuroticism, reflection subtype of rumination, although one study found no depression, and emotion-based coping strategies (31), all of evidence on the mediating effect of brooding between childhood which have been associated with rumination (1). maltreatment and adolescent internalizing or externalizing In our present study, we investigated the associations of psychopathology (7). The relationship between childhood rumination with two regulatory SNPs from the two ends of maltreatment and reflection is contradictory in itself, since two the HTR2A gene. In addition to the promoter methylation site studies suggest their positive association (4, 6), but another one rs6311 (25), we chose the miRNA binding site rs3125 (https:// suggests no association between them (5). In contrast, brooding snpinfo.niehs.nih.gov/snpinfo/snpfunc.html) residing in the level is consistently predicted by childhood maltreatment (4, 6) 3′ UTR (untranslated region), the other regulatory side of the and its all forms except for physical neglect (5). gene, according to hg19 database of the UCSC Genome Browser The moderating role of the5-HTTLPR length polymorphism (https://genome.ucsc.edu/). Rs3125 is supposed to bind miR- of the serotonin transporter gene in the effect of childhood 1270, miR-1304, miR-202, miR-539, and miR-620 (https:// maltreatment on rumination (8) indicates that the serotonin snpinfo.niehs.nih.gov/cgi-bin/snpinfo/mirna.cgi?2_rs3125). system seems to play a relevant role in rumination. In addition, It can be a tag SNP, representing its haploblock that consists of tryptophan depletion in humans provokes cognitive inflexibility two SNPs (Figure 1), and it has been associated with depression and a deficit in inhibitory control 9( ), both of which have been among cardiac patients (32). We tested the main effect of these associated with rumination (10, 11), further supporting the two SNPs, and their interactions with childhood adversity, on involvement of serotonin function in rumination. rumination and its two subtypes: brooding and reflection. In Among elements of the serotonin system, the neocortical case of finding a significant effect, we tested whether or not it is serotonin 2A (5-HT2A) receptor appears to be a promising additional mediated or moderated by depression. We hypothesize to reveal candidate with regard to rumination, since it mediates the effect both main effects and interaction effects of the SNPs, based on of tryptophan depletion on response inhibition (12), and it has the abundant main effect findings ofHTR2A and the interaction also shown alterations in radioligand binding and functional effect literature of5-HTTLPR . As supplementary analyses, we regulation by messenger RNA levels and by protein kinase A ran the same models with 5-HTTLPR. activity in the prefrontal cortex in several psychiatric disorders

(13). Moreover, frontal 5-HT2A binding has been associated with dysfunctional attitudes among patients in a major depressive METHODS episode (14, 15), and 5-HT binding in the left dorsal prefrontal 2A Our study, carried out in accordance with the Declaration of cortex has shown a positive correlation with the anticipatory Helsinki, and being part of the NewMood (New Molecules in worry subscale of harm avoidance (16). Dysfunctional attitudes Mood Disorders) study funded by the European Union (Sixth and worry are both related to rumination (1, 10), consistent with Framework Program of the EU, LSHM-CT-2004-503474), was a link between 5-HT and rumination. 2A approved by the Scientific and Research Ethics Committee of the Early life stress can affect gene expression through epigenetic Medical Research Council, Budapest, Hungary, and by the North mechanisms including DNA methylation and microRNA Manchester Local Research Ethics Committee, Manchester, (miRNA) expression (17). Therefore, in investigating the United Kingdom. link between rumination-related phenotypes, childhood maltreatment and functioning of 5-HT2A, single nucleotide polymorphisms (SNPs) facilitating epigenetic regulation of the Participants HTR2A gene encoding the receptor appear to be promising Subjects provided written informed consent, and they were targets. Such regulatory SNPs have extensively been investigated recruited through advertisements and general practices from in the background of rumination-related phenotypes. Increased Budapest, Hungary, and through advertisements, general HTR2A gene expression has been inconsistently linked to either practices, and a website from Greater Manchester, UK. They the T allele of the methylation site T102C (rs6313) (18) SNP of received nothing for participation. exon-1 in the cortex (15), or to its C allele in the frontal lobe (19). There were 1,501 adults (18–60 years old) from Budapest Nevertheless, TC heterozygotes perform worse on the Wisconsin (N = 470) and Manchester (N = 1,031) that provided self- Card Sorting Task (WCST) than both homozygote groups report questionnaire data about sex, age, and rumination,

Frontiers in Psychiatry | www.frontiersin.org 2 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

FIGURE 1 | Position of rs3125 within the HTR2A gene on chromosome 13 (https://genome.ucsc.edu/, build hg19), and among tag single nucleotide polymorphisms (SNPs) of the gene in the HapMap CEU population (https://snpinfo.niehs.nih.gov/snpinfo/snptag.html). CEU = a population with Northern and Western European ancestry (https://www.sanger.ac.uk/resources/downloads/human/hapmap3.html); LD = linkage disequilibrium between each pair of SNPs, measured with r2; MAF = minor allele frequency. which reported to be of European white ethnic origin and were Five items of RRS belong to the brooding subscale, and five items to successfully genotyped for rs3125. None of them reported having the reflection subscale, and scores on the two subscales add up to had any relative participating in the study. Among these included the rumination score. Brooding and reflection scores have a Pearson participants, rs6311 was successfully genotyped in 469 subjects correlation of r = 0.487 (N = 1,501; p < 0.00001) in the combined from Budapest and 1,017 from Manchester, and childhood sample, r = 0.308 (N = 470; p < 0.00001) in the Budapest subsample, adversity measure was provided by 468 subjects from Budapest and r = 0.521 (N = 1,031; p < 0.00001) in the Manchester subsample. and 1,030 from Manchester. Data on both rs6311 and childhood We used depression items plus the additional items from adversity were available in 467 subjects from Budapest, and 1,016 the Brief Symptom Inventory (BSI) (33) to measure current from Manchester. Supplementary Figure 1 displays a flowchart depressive symptoms. Each of the rumination scores and the BSI on inclusion criteria and data availability. depression score was calculated as a weighted score: the sum of item scores divided by the number of completed items. Lifetime depression was measured by a question in the background Phenotypic Measures questionnaire, and had been validated previously with face-to- We measured rumination and its two subscales, brooding and face diagnostic interviews within a subpopulation, yielding a reflection, with the 10-item Ruminative Responses Scale (RRS) 2( ). 91.7% sensitivity and 89.8% specificity 34( ).

Frontiers in Psychiatry | www.frontiersin.org 3 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

Our childhood adversity measure (provided as Supplementary the main effect models, and, in case of interaction models, 2 Data) was based on the Childhood Trauma Questionnaire (CTQ) assuming an RGE = 0.5% for the gene–by–environment (GxE) 2 (35), but is different from that in the specific items. Childhood interaction, RG = 0% for the genetic main effect, and based on 2 adversity score means the sum score of four items regarding coefficients of determination with childhood adversity, anE R = emotional and physical abuse and neglect, and two items about 0.066 for rumination, 0.0756 for brooding and 0.0276 or 0.0282 parental loss. We had validated this short childhood adversity (as appropriate for the respective sample size) for reflection. measure previously with the 28-item CTQ within a subpopulation, For significant findings among the 36 primary tests, we tested yielding a significant Pearson r = 0.75 (p < 0.001) (34). possible mediating effects in secondary (post hoc) analyses as follows. First, we built the same model as that of the significant Genotyping finding, with logistic regression on lifetime depression and linear regression on BSI depression as the outcome. Second, we ran the By a genetic saliva sampling kit, subjects provided buccal same model again, on the rumination variable as the outcome mucosa cells personally or by post, from which genomic DNA and including both depression variables as additional covariates. was extracted according to Ref. (36). Rs3125 and rs6311 were Third, we ran the model on both depression variables separately, genotyped with the Sequenom MassARRAY technology (www. including the rumination variable as an additional covariate. sequenom.com, Sequenom, San Diego, CA, USA). Genotyping To control for the overfitting of these models due to the several of 5-HTTLPR has been detailed in one of our previous papers covariates, 100,000 permutations were run on the relevant term in (37). All laboratory works were performed under the ISO each mediation model. To speed up analyses, permutation was run 9001:2000 quality management requirements, and it was blinded in Plink v1.90b6.9 (4th March, 2019; https://www.cog-genomics. with regard to phenotype. org/plink/1.9/). A label-swapping, max(T) permutation procedure was applied, yielding an empirical p-value for the regression term. Statistical Analyses Besides mediating, moderating effects of depression were We used Plink v1.07 (http://zzz.bwh.harvard.edu/plink/) to also tested post hoc for the significant findings, by running the calculate Hardy–Weinberg equilibrium and allele frequencies, same model separately in those who reported lifetime depression and, with the aid of individually written scripts in R (38), to run (ever-depressed) and in those who did not (never-depressed). regression models. SPSS25 was used to perform χ2 or t tests, Significant findings were post hoc tested whether holding as appropriate, for descriptive statistics, and univariate general true in the separate Budapest and Manchester subsamples, linear models for visualization purposes in the figures. In these by the same models described above but without population as general linear models, childhood adversity score was grouped a covariate. into three categories: low (0–3); medium (4–6); and high (7 or To test effects of the 5-HTTLPR length polymorphism residing more). Possible childhood adversity score ranges from 0 to 18, within the promoter region of the serotonin transporter gene, and in our study from 0 to 16. The same grouping to low, medium, its main effects and interaction effects with childhood adversity and high scores had been applied in our previous study (39). In were tested within the combined sample in a manner similar to the general linear models run to visualize interaction effects, HTR2A testing, as supplementary analyses. covariates were main effects of the respective SNP and childhood In all post hoc analyses, and also in results of descriptive statistics adversity, and the outcome variable had been controlled for the and supplementary analyses, the threshold for significance wasp ≤ covariates detailed below, in a previous general linear model. 0.05, and for trend, it was p ≤ 0.10. In our Plink linear regression models population, sex and age were covariates in all the analyses, and in case of each rumination subscale as the outcome, the other subscale was also a covariate. RESULTS When testing an SNP × childhood adversity interaction effect, main effects of both the SNP and childhood adversity were Descriptive Statistics included as covariates in the model. For frequencies or means of the variables, see Table 1 and As primary analyses, 36 regression equations were run in the Supplementary Table 1. We can see that the two subsamples Budapest + Manchester combined sample: with either of the two significantly differ from each other in all variables except for SNPs as predictors, on rumination, brooding and reflection as the rs6311 and 5-HTTLPR genotype frequencies. outcome variable, testing either a main effect or an interaction Both HTR2A SNPs are in Hardy–Weinberg equilibrium effect of the SNP, in additive, dominant, and recessive models. in the combined sample and in the Manchester and Budapest QVALUE v1.0 (40) was used to calculate q-values of false subsamples (rs3125 has a p = 0.909 in the combined sample, discovery rate (FDR) for the p-values of these 36 tests, without p = 0.616 in Budapest, and p = 0.607 in Manchester; and rs6311 robust method, with a 0 to 0.99 range of λ (by 0.05), and a yields a p = 0.163 in the combined sample, p = 0.704 in Budapest, bootstrap method to estimate the overall proportion of true null and p = 0.051 in Manchester). For rs3125, C is the minor allele, hypotheses, π0. We consider results with a q-value of ≤0.05 as with an allele frequency of 0.1289. For rs6311, T is the minor significant after correction for multiple testing. allele, yielding an allele frequency of 0.4078. For these primary tests, power calculation was carried out Table 2 demonstrates that there is a significant gene-environment with Quanto v1.2 (http://biostats.usc.edu/Quanto.html), at a correlation in case of rs3125 and childhood adversity score in the 2 type I error rate of 0.05, assuming an RG = 1% for the SNP in combined sample and in Manchester.

Frontiers in Psychiatry | www.frontiersin.org 4 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

TABLE 1 | Descriptive statistics for the combined Budapest + Manchester sample and the two subsamples.

Budapest + Manchester Budapest Manchester Difference between Budapest and Manchester

Frequency % Frequency % Frequency % χ2 p

Sex Male 371 24.7% 88 18.7% 283 27.4% 13.209 0.0003 Female 1,130 75.3% 382 81.3% 748 72.6% Lifetime Not reported 824 54.9% 370 78.7% 454 44.0% 156.889 <0.00001 depression Reported 677 45.1% 100 21.3% 577 56.0% HTR2A rs3125 CC 24 1.6% 6 1.3% 18 1.7% 8.678 0.013 CG 339 22.6% 85 18.1% 254 24.6% GG 1,138 75.8% 379 80.6% 759 73.6% HTR2A rs6311 TT 234 15.7% 83 17.7% 151 14.8% 2.589 0.274 TC 744 50.1% 223 47.5% 521 51.2% CC 508 34.2% 163 34.8% 345 33.9%

Mean S.E.M. Mean S.E.M. Mean S.E.M. t p Age 32.823 0.2747 30.315 0.4925 33.967 0.3249 −6.244 <0.00001 Rumination score 2.174 0.0151 1.986 0.0209 2.259 0.0192 −9.601 <0.00001 Brooding score 2.197 0.0178 1.954 0.0250 2.308 0.0224 −10.574 <0.00001 Reflection score 2.150 0.0172 2.019 0.0267 2.210 0.0217 −5.543 <0.00001 Childhood adversity score 3.392 0.0895 2.801 0.1365 3.660 0.1136 −4.838 <0.00001 BSI depression score 0.900 0.0244 0.540 0.0283 1.063 0.0319 −12.268 <0.00001

χ2, Pearson χ2; S.E.M., standard error of mean; BSI, Brief Symptom Inventory.

TABLE 2 | Effect of each HTR2A single nucleotide polymorphism (SNP) as predictor, for childhood adversity as outcome, in linear regression models.

Additive model Dominant model Recessive model

Beta P-value Beta P-value Beta P-value rs3125 Budapest + Manchester 0.422 0.024 0.544 0.008 −0.338 0.630 Budapest 0.182 0.561 0.275 0.424 −0.677 0.575 Manchester 0.494 0.032 0.629 0.014 −0.286 0.739 rs6311 Budapest + Manchester 0.021 0.869 0.048 0.797 −0.006 0.979 Budapest −0.122 0.529 −0.483 0.090 0.338 0.343 Manchester 0.102 0.547 0.302 0.205 −0.174 0.584

Sex and age (and in the combined sample also population) were covariates. The minor, effect allele is C for rs3125, and T for rs6311. Significant findings are marked with bold.

Among the 36 primary analysis models, those that test only Moderating Role of Depression in the the main effect of the SNP, without interaction term, have a Effect of the Investigated HTR2A Variants 97.16% and a 97.28% power to detect it in case of rs6311 and on Rumination Phenotypes rs3125, respectively. In case of testing the interaction effect with The rs6311 × childhood adversity interaction on rumination in childhood adversity, rs6311 has 80.55% power for rumination, an additive model remains significant both in ever-depressed 80.95% for brooding, and 78.98% for reflection. Rs3125, in (N = 670; β = 0.015; p = 0.045) and in never-depressed (N = 813; interaction with childhood adversity, has 80.94% power for β = 0.019; p = 0.028) participants, indicating that depression rumination, 81.34% for brooding, and 79.36% for reflection. history does not moderate this effect. These findings indicate sufficient power to detect the tested Similarly, the rs3125 × childhood adversity interaction on genetic effects. brooding remains significant in both ever-depressed (N = 676; additive β = 0.026; p = 0.022; dominant β = 0.029; p = 0.023) and never-depressed (N = 822; additive β = 0.032; p = 0.034; Effects of HTR2A rs3125 and rs6311 dominant β = 0.033; p = 0.033) participants. on Rumination and Its Subtypes Among the 36 equations (Table 3), only the rs6311 × childhood adversity interaction on rumination in an additive model (Figure Mediating Role of Depression in the 2), and the rs3125 × childhood adversity interaction on brooding Found Effects in both an additive and a dominant (Figure 3) model, proves to To test the possible mediating role of depression in the rs6311 × be significant after correction for multiple testing. childhood adversity interaction effect on rumination and, vice

Frontiers in Psychiatry | www.frontiersin.org 5 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

TABLE 3 | Effect of each HTR2A SNP as predictor, for each rumination variable as outcome, in linear regression models.

Outcome SNP Additive model Dominant model Recessive model

Beta P-value Q-value Beta P-value Q-value Beta P-value Q-value

Rumination rs3125 0.041 0.180 0.114 0.061 0.068 0.085 −0.133 0.245 0.116 rs6311 0.001 0.953 0.212 0.011 0.724 0.176 −0.014 0.727 0.176 Brooding rs3125 0.010 0.747 0.176 0.014 0.686 0.176 −0.020 0.871 0.199 rs6311 0.020 0.367 0.124 0.030 0.348 0.124 0.019 0.640 0.176 Reflection rs3125 0.035 0.278 0.116 0.053 0.132 0.114 −0.127 0.291 0.116

Main effect of Main effect SNP rs6311 −0.019 0.400 0.124 −0.018 0.570 0.166 −0.034 0.404 0.124 Rumination rs3125 0.011 0.173 0.114 0.011 0.203 0.116 0.020 0.582 0.166 rs6311 0.015 0.013 0.035 0.017 0.046 0.073 0.022 0.042 0.073 Brooding rs3125 0.028 0.001 0.006 0.030 0.001 0.006 0.036 0.345 0.124 rs6311 0.008 0.227 0.116 0.010 0.282 0.116 0.010 0.394 0.124 Reflection rs3125 −0.015 0.091 0.091 −0.017 0.075 0.085 −0.013 0.729 0.176

SNP × childhood adversity interaction rs6311 0.009 0.147 0.114 0.010 0.286 0.116 0.016 0.186 0.114

Population, sex, and age were additional predictors in all models. In case of brooding or reflection as outcome, the other subscale was also a predictor. In the interaction models, main effects of both the respective SNP and childhood adversity were included as additional predictors. Findings having a q ≤ 0.05, thus surviving the correction for multiple testing, are marked with bold. The minor, effect allele is C for rs3125, and T for rs6311. SNP, single nucleotide polymorphism.

FIGURE 2 | Interaction effect of childhood adversity and HTR2A rs6311 genotype on rumination score in a general linear model performed with visualization purposes. versa, the possible mediating role of rumination in the same significant but with almost the same effect sizeN ( = 1,482; β = 0.014; genetic effect on depression, the following prerequisites have to p = 0.005; empirical p = 0.005) as can be seen without controlling for be met. Depression phenotypes have to show a significant positive depression (Table 3). This strengthening of significance may be due correlation with rumination, and have to be associated with the to the confounding effect of depression in the common variance of rs6311 × childhood adversity interaction term in the same direction rumination and rs6311 × childhood adversity. as rumination does. Rumination indeed shows a positive association Prerequisites of testing the mediating role of depression in the with lifetime depression (N = 1,483; t = −18.304; p < 0.00001; with significant rs3125 × childhood adversity effect on brooding are met. rumination means of 1.944 in the never-depressed and 2.451 in Brooding has a significant positive association with both lifetime the ever-depressed group) and with BSI depression (N = 1,482; depression (N = 1,498; t = −18.896; p < 0.00001; with means of 1.920 Pearson r = 0.570; p < 0.00001). However, the rs6311 x childhood in the never-depressed and 2.534 in the ever-depressed group) and adversity additive model does not yield a significant effect on either BSI depression (N = 1,497; Pearson r = 0.618; p < 0.00001). The lifetime depression (N = 1,483; odds ratio (OR) = 1.022; p = 0.419), rs3125 × childhood adversity interaction effect that has been found or BSI depression (N = 1,482; β = −0.001; p = 0.894), therefore we significant on brooding, is a trend on both lifetime depression (N = cannot test the possible mediating role of depression in this genetic 1,498; additive OR = 1.081; p = 0.070; dominant OR = 1.087; p = effect on rumination. Nevertheless, including both depression 0.064) and BSI depression (N = 1,497; additive β = 0.024; p = 0.067; phenotypes as covariates, the rs6311 × childhood adversity dominant β = 0.026; p = 0.060). All these associations enabled us interaction on rumination in an additive model becomes more to test mediating effects. Including both depression phenotypes as

Frontiers in Psychiatry | www.frontiersin.org 6 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

FIGURE 3 | Interaction effect of childhood adversity and HTR2A rs3125 genotype on brooding score in a general linear model performed with visualization purposes.

additional covariates, the rs3125 × childhood adversity interaction p = 0.309), but it is significant in Manchester (β = 0.015;p = effect on brooding remains significant at a nominalp ≤ 0.05 0.050) (Figure 4). level in both additive (β = 0.018; p = 0.015; empirical p = 0.015) The rs3125× childhood adversity effect on brooding can be and dominant (β = 0.019; p = 0.017; empirical p = 0.017) models. replicated, because it is significant in both Budapest (additive β = However, including brooding as an additional covariate, the effect 0.042; p = 0.026; dominant β = 0.042; p = 0.029) and Manchester of the rs3125 × childhood adversity interaction term considerably (additive β = 0.024; p = 0.016; dominant β = 0.026; p = 0.016) weakens on both lifetime depression (additive OR = 1.035; p = 0.449; (Figure 5). empirical p = 0.453; dominant OR = 1.038; p = 0.430; empirical p = 0.431) and BSI depression (additive β = 0.005; p = 0.660; empirical Effects of 5-HTTLPR on Rumination p = 0.659; dominant β = 0.006; p = 0.600; empirical p = 0.600). and Its Subtypes Supplementary Table 2 demonstrates that 5-HTTLPR is not Replicability of Significant Findings in associated with childhood adversity, entailing that there is no the Separate Budapest and Manchester gene-environment correlation in its case. Subsamples Supplementary Table 3 shows that 5-HTTLPR is not The rs6311× childhood adversity additive model on rumination associated with rumination, brooding or reflection, either in is not replicable, since it is not significant in Budapest (β = 0.009; main effect or in interaction with childhood adversity.

FIGURE 4 | Interaction effect of childhood adversity and HTR2A rs6311 genotype on rumination score in a general linear model performed with visualization purposes in the Budapest (A) and Manchester (B) subsamples.

Frontiers in Psychiatry | www.frontiersin.org 7 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

FIGURE 5 | Interaction effect of childhood adversity and HTR2A rs3125 genotype on brooding score in a general linear model performed with visualization purposes in the Budapest (A) and Manchester (B) subsamples.

DISCUSSION 5-HTTLPR and rumination itself, a possible mediating link between the detrimental impact of childhood maltreatment We have demonstrated that both of two HTR2A polymorphisms and serotonin transporter gene function may be regulation related to two distinct epigenetic regulatory mechanisms exert by epigenetic mechanisms. Epigenetic alterations, which are a significant effect on current adult rumination as a function of heritable and environmentally modifiable, denote reversible childhood stress, independently of current and lifetime depression modifications to the genome, and they engender alterations status. Both the methylation site rs6311 and the miRNA in gene expression patterns in a cell-type specific manner binding site rs3125 appear to contribute to the endophenotypic (41). Early life stress has been proposed to have long-term manifestation of rumination, and these findings also support the consequences on brain structure and mental health outcomes need to consider the role of childhood stress when considering via epigenetic mechanisms including methylation and miRNA endophenotypes. Moreover, the impact of the rs3125 × childhood expression, which were found to influence mental health adversity interaction on brooding could be replicated in Budapest phenotypes and serotonin transporter expression, respectively and Manchester, remained significant when controlling for (17). An exposure during development may impact epigenetic depression, and also fully mediated the weak effect of the same states more broadly than later exposure (42), and especially interaction term on depression. Thus, rs3125 and brooding show miRNAs can totally switch expression during development but a stronger contribution to the endophenotypic manifestation of fine-tune it in adult tissues 43( ). In addition to former (but rumination phenotypes than rs6311 and a broader measure of not present) findings with 5-HTTLPR, our present results with rumination that encompasses the reflection subtype in addition HTR2A also underline the relevance of the serotonin system to the brooding subtype. Rs3125 and childhood adversity in the long-lasting effects of childhood stress that may be may also convey a transdiagnostic relevance to the brooding transmitted by epigenetic mechanisms. endophenotype. In the promoter region of HTR2A, the cytosine at position -1439 can only be methylated in case of a G allele (or C allele if measured on the complementary, positive strand of DNA, as HTR2A Effect on Adulthood Rumination in our study) at the adjacent -1438 A/G (rs6311) polymorphism Phenotypes Depends on the Level of (19, 25, 44). Although methylation level of the promoter region Childhood Adversity, Independently of of HTR2A was inversely related to the transient phenotype History of Depression of antipsychotic use in the frontal lobe of schizophrenic and Contrary to former findings concerningHTR2A and bipolar patients (19), its methylation in the placenta can entail rumination-related phenotypes (20, 21, 31), in our study, we long-term impacts on psychiatric phenotypes (44, 45). Previous found no main effect of any SNP in itself on rumination, but contradictory results with rs6311 (26, 29–31) can be resolved only an interaction with childhood adversity proved to be by considering the moderating role of stress level, but recent significant, similarly to the 5-HTTLPR × childhood maltreatment stress has been suggested as much important as early stress in interaction effect on rumination reported by Antypa and these terms. TT genotype denoted a risk for depression in case Van der Does (8). In line with this, our former results with of a high childhood adversity level (46), and for a reduced heart 5-HTTLPR and childhood adversity have also demonstrated rate variability only in case of a high level of recent stress (47). the role of early tuning of the serotonin system in adulthood Heart rate variability has an inverse association with brooding stress reactivity leading to depression (39). Although our (48), so our present results (see Figure 2) corroborate these present study did not find any significant association between former findings with another type of stress. As the T allele of

Frontiers in Psychiatry | www.frontiersin.org 8 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination rs6311 enhances expression (15, 26–28), and in accordance with seems multifaceted and can be further deconstructed to even that tryptophan depletion impairs response inhibition via an simpler endophenotypes (59). Brooding may be its more useful increased 5-HT2A density within the right inferior frontal gyrus subtype in primary and secondary depression prevention than (12), we can hypothesize that a genetically heightened expression the broader measure of rumination that includes also reflection. of HTR2A may make the level of rumination more dependent Indeed, reflection has not shown any association with any of the on 5-HT2A-mediated serotonin transmission, thus more sensitive HTR2A polymorphisms in our present results. to environmental impacts affecting the serotonin system, such as Brooding may be a more useful endophenotype first because tryptophan depletion or stress. the rs3125 × childhood adversity interaction on brooding is Similarly, former results with the miRNA binding site rs3125 replicable in Budapest and Manchester, while the rs6311 × were inconclusive on bipolar disorder when stress level was childhood adversity interaction on rumination is significant not taken into account (49). On the other hand, the C allele only in Manchester but not in Budapest. Robustness of GxE appeared to increase risk of depression in cardiac patients (32) (gene–by–environment) results on brooding is remarkable also which can be considered a stressed group, corroborating our because participants from Manchester have higher scores on results also with the C allele as a risk variant for brooding only in childhood adversity and brooding, have a lower frequency of case of high childhood stress (Figures 3 and 5). Rs3125 can bind rs3125 GG genotype, and are more depressed than participants to five different miRNAshttps://snpinfo.niehs.nih.gov/cgi- ( from Budapest (Table 1). Since our subsamples are not at all bin/snpinfo/mirna.cgi?2_rs3125) (50). Among them, miR-539 representative for the two populations in the variables of interest, bound by the G allele of rs3125 showed a reduced expression in further investigation is needed regarding the relative cross- anterior cingulate cortex (ACC) in an animal model of chronic population robustness of these genetic associations with brooding neuropathic pain (51). Although ACC has demonstrated and the broader measure of rumination compared to each other. a negative association in volume and resting state activity Second, because the effect of HTR2A on brooding seems with rumination (52), and 5-HT2A binding in ACC has been more relevant in depression than its effect on more broadly negatively related to treatment resistance in major depression measured rumination. While the rs6311 × childhood adversity (53), the exact role of rs3125 in HTR2A expression and the role interaction term is not associated with depression at all, the of miRNA-regulated HTR2A expression in brooding have to rs3125 × childhood adversity interaction term has a trend effect be elucidated in the future. It should also be clarified whether on depression, which disappears if controlling for brooding. childhood or recent timing of stress matters in the effect of Brooding, however, remains associated with rs3125 × childhood rs3125 on brooding. adversity even if controlling for depression. All of these results Both HTR2A × childhood adversity interaction effects point to a stronger association of rs3125 with brooding than with were replicable separately in the ever-depressed and the depression and suggest that rs3125 contributes to brooding as an never-depressed group, contributing to the endophenotypic endophenotype (59) that confers a risk for depression. manifestation of rumination. Namely, an endophenotype, The investigatedHTR2A genotypes exert an effect only on by definition, should reside on the causal pathway between the broad measure of rumination and its brooding subtype but genetics and the disorder (54, 55), and rumination is not only not on reflection. Brooding, being a more maladaptive subtype augmented by a depressive episode even after recovery 56( , 57), of rumination than reflection (2), may be a closer construct to but it also predicts future onset of depression in never-depressed those phenotypes previously having shown an association with individuals (58). Our results point to the role of genetics in these 5-HT2A or HTR2A: response inhibition deficit 12( ), perseverative associations and thus complete the picture with endophenotypic errors on WCST (21), worry (16), dysfunctional attitudes (14, 15), features, highlighting that HTR2A affects rumination not only neuroticism and emotion-based coping strategies (31). Moreover, in or after a depressive episode, but equally in those who have our genetic findings with HTR2A × childhood adversity on not yet developed or will never develop the disorder. Since high rumination, brooding, and depression underline and expand rumination constitutes a risk for depression, screening never- previous results that only brooding but not reflection is important depressed people based on HTR2A rs3125 and rs6311 genotypes in the depressogenic effect of childhood maltreatment 4( –6). and levels of childhood adversity and rumination may once Further research is needed to clarify the origin of difference be part of a comprehensive picture in the primary prevention between miRNA binding and methylation of HTR2A in of depression. Similar screening in ever-depressed people contributing to the endophenotypic manifestation of either could similarly aid relapse prevention as well as decision on brooding or the broader measure of rumination. Although pharmacotherapeutic and psychotherapeutic intervention. considerable evidence underscores the interconnectedness of miRNA function and DNA methylation in gene regulation (60), also pointing to the precedence of miRNA functioning over Effect of HTR2A rs3125 × Childhood DNA methylation in neuronal differentiation (61) and possibly Adversity on Brooding, a Possible in haloperidol effects (62), the exact relationship of these two Transdiagnostic Endophenotype types of epigenetic regulation regarding particularly HTR2A Spanning European Populations expression needs to be clarified. Although rumination score has shown endophenotypic features Nevertheless, we can suggest that rs3125 can be a more useful in our study, in that its association with rs6311 can be replicated biomarker in primary and secondary depression prevention both in the ever-depressed and the never-depressed group, it also than rs6311.

Frontiers in Psychiatry | www.frontiersin.org 9 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

The fact that the rs3125× childhood adversity interaction effect stress and rumination. The same concept can then be applied on brooding explains the same interaction effect on depression in hypothesis-free genome-wide investigations, going beyond but is not explained by depression, implies that it goes beyond candidate genes. depression and may play a role in the potential transdiagnostic Rumination was measured only by RRS, a self-report relevance of the brooding endophenotype. It may be part of questionnaire that asks people to rate themselves related to when the endophenotype conveying a risk for the disorders having they feel depressed. Other rumination measurements would also been linked to both rumination and HTR2A, such as alcohol be worth investigations with regard to HTR2A. abuse (63–65), binge eating (66, 67) and obsessive-compulsive Stress was defined only as childhood stress. A more detailed disorder (68–70). Transdiagnostic relevance of specifically the picture would be gained by targeting the possible role of other brooding subtype of rumination has already been suggested in types of stress, such as recent stress or medical conditions. obsessive-compulsive disorder and generalized anxiety disorder Our study population is limited to European white among unipolar depressed patients (68). Usefulness of HTR2A participants. To strengthen clinical relevance of our present rs3125, childhood adversity and brooding in the primary and results, future studies should confirm the same associations in secondary prevention of all these disorders should be revealed other ethnicities. in future studies. Future studies should measure actual epigenetic markers that are supposed to mediate between the revealed GxE interactions Limitations and rumination phenotypes. Our study has several limitations which must be stated. Our statements about the effect of childhood adversity on adulthood Conclusions and Future Directions rumination and the moderating role of genetic variants in Both of the investigated genetic variations involved in this effect could be proven only in case of a longitudinal study transmitting two distinct epigenetic regulatory mechanisms, design. However, our design being cross-sectional, we can draw promoter DNA methylation and miRNA binding in the 3′ conclusions only about associations between childhood stress UTR, acting on HTR2A gene, contribute to the endophenotypic and adulthood phenotype. A longitudinal design could similarly manifestation of rumination, in that their effects can be aid in clarifying the causal roles of rumination and depression in detectable not only in depression but before or without the each other. emergence of depression. Potentials of targeting HTR2A Furthermore, childhood stress was assessed retrospectively, genetics in depression prevention deserve further studies. Our and only by self-report of our subjects, but not ascertained by results on action of HTR2A in rumination phenotypes also other informants. Thus assessment of childhood adversity is underscore the need to include childhood adversity assessment subject to memory, recall bias and also state-dependent recall, and in these possible prevention strategies, and, more broadly, to could be biased by voluntary distortions related to psychiatric and include stress or other environmental factors in considering personality disorders. Similarly, lifetime depression assessment endophenotypes, especially in case of polymorphisms conveying was based on self-report but not corroborated by actual previous epigenetic impacts. Brooding seems a more promising candidate disease history. endophenotype in mediating vulnerability to depression than Since we found a gene-environment correlation between rumination measured more broadly, since its association rs3125 and childhood adversity both in the combined sample with HTR2A can be replicated across two different European and in Manchester, it was crucial to replicate our rs3125 populations, and mediates the same genetic association with interaction findings in Budapest, and to include main effects of depression. The effect of rs3125 on brooding, depending on gene and environment as covariates in these interaction models. childhood adversity level, may have a wider transdiagnostic Our rs3125 × childhood adversity finding on brooding could be relevance for other disorders in which brooding may be replicated also in Budapest, underlining that it was not, or not important such as obsessive-compulsive disorder. only due to the gene-environment correlations. In the present study, we did not consider the behavior of individual items with respect to the applied scale scores, DATA AVAILABILITY STATEMENT but treated them as equivalently weighted in adding up to The datasets generated for this study are available on request to the respective score. In the future, item-response analyses the corresponding author. should argue for or against this equivalent ponderation of the individual items. Another limitation of our study is the low number of SNPs ETHICS STATEMENT within the investigated gene. Tagging HTR2A with more SNPs, or broadening our scope to rare variants, structural variants, Our study, carried out in accordance with the Declaration of copy number variants or other length polymorphisms, besides Helsinki, was approved by the Scientific and Research Ethics variants with a more diverse spectrum of annotation types (such Committee of the Medical Research Council, Budapest, as amino acid change as an additional type of consequence Hungary, and by the North Manchester Local Research Ethics on gene functioning), will be able to provide a deeper insight Committee, Manchester, United Kingdom. Subjects provided into the role of HTR2A gene in the association of childhood written informed consent.

Frontiers in Psychiatry | www.frontiersin.org 10 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

AUTHOR CONTRIBUTIONS ACKNOWLEDGMENTS

JD, GB, IA, GJ, RE, and XG designed the study. GJ and XG We thank the Cheadle and the Heaton Mersey Medical Practices performed data collection. NE, PP, and DB undertook statistical for recruitment. We further thank Diana Chase, Darragh Downey, analyses. NE managed the literature search and wrote the first Kathryn Lloyd-Williams, Emma J. Thomas, and Zoltan G. Toth draft of the manuscript. All authors contributed to manuscript for recruitment and data acquisition. We thank Krisztina Mekli revision, read, and approved the submitted version. and Hazel Platt for genotyping. We also thank Fanni Bákonyi for her help in figure preparation. FUNDING AUTHOR’S NOTE This study, as part of NewMood, was supported by the Sixth Framework Program of the European Union (LSHM-CT-2004-503474). Preliminary results of this work were presented by Nora Eszlari on Moreover, it was supported by the Hungarian Academy of Sciences the ECNP Workshop for Junior Scientists in Europe, 17–20 March (MTA-SE Neuropsychopharmacology and Neurochemistry 2016, Nice, France. It was published as an abstract in European Research Group), by the Hungarian Brain Research Program (grants Neuropsychopharmacology 26, S77–S78, with the title “Brooding KTIA_13_NAPA-II/14 and 2017-1.2.1-NKP-2017-00002, and with subtype of rumination is modulated by the interplay between the Hungarian National Development Agency, the Hungarian serotonin receptor 2A gene and childhood adversity” (72). Academy of Sciences and Semmelweis University, grant KTIA_ Results of this work appeared first and only in Nora Eszlari’s NAP_13-2-2015-0001, MTA-SE-NAP B Genetic Brain Imaging PhD dissertation (71). It is in line with the policy of Semmelweis Migraine Research Group), by the National Development Agency University, and the dissertation can be accessed online (http:// (KTIA_NAP_13-1-2013-0001), by the New National Excellence semmelweis.hu/wp-content/phd/phd_live/vedes/export/ Program of The Ministry of Human Capacities (grants ÚNKP-16-3; eszlarinora.d.pdf). ÚNKP-17-3-III-SE-2; ÚNKP-17-4-I-SE-8 and ÚNKP-18-4-SE-33), by TAMOP-4.2.1.B-09/1/KMR-2010-0001, and by the National Institute for Health Research Manchester Biomedical Research SUPPLEMENTARY MATERIAL Centre. XG is recipient of the Janos Bolyai Research Fellowship of the Hungarian Academy of Sciences. None of the sponsors had any role The Supplementary Material for this article can be found online at: in study design, data collection, analysis or interpretation, writing https://www.frontiersin.org/articles/10.3389/fpsyt.2019.00394/ the report, or in the decision to submit the paper for publication. full#supplementary-material

REFERENCES 9. Logue SF, Gould TJ. The neural and genetic basis of executive function: attention, cognitive flexibility, and response inhibition. Pharmacol Biochem 1. Nolen-Hoeksema S, Wisco BE, Lyubomirsky S. Rethinking rumination. Behav (2014) 123:45–54. doi: 10.1016/j.pbb.2013.08.007 Perspect Psychol Sci (2008) 3:400–24. doi: 10.1111/j.1745-6924.2008.00088.x 10. Larsen BA, Christenfeld NJ. Cardiovascular disease and psychiatric 2. Treynor W, Gonzalez R, Nolen-Hoeksema S. Rumination reconsidered: comorbidity: the potential role of perseverative cognition. Cardiovasc a psychometric analysis. Cogn Ther Res (2003) 27:247–59. doi: Psychiatry Neurology (2009) 2009:791017. doi: 10.1155/2009/791017 10.1023/A:1023910315561 11. Whitmer AJ, Gotlib IH. An attentional scope model of rumination. Psychol 3. Spasojević J, Alloy LB. Who becomes a depressive ruminator? Developmental Bull (2013) 139:1036–61. doi: 10.1037/a0030923 antecedents of ruminative response style. J Cogn Psychother (2002) 16:405– 12. Macoveanu J, Hornboll B, Elliott R, Erritzoe D, Paulson OB, Siebner H, 19. doi: 10.1891/jcop.16.4.405.52529 et al. Serotonin 2A receptors, citalopram and tryptophan-depletion: a 4. Raes F, Hermans D. On the mediating role of subtypes of rumination in multimodal imaging study of their interactions during response inhibition. the relationship between childhood emotional abuse and depressed mood: Neuropsychopharmacology (2013) 38:996–1005. doi: 10.1038/npp.2012.264 brooding versus reflection.Depress Anxiety (2008) 25:1067–70. doi: 10.1002/ 13. Aznar S, Klein AB. Regulating prefrontal cortex activation: an emerging role da.20447 for the 5-HT(2)A serotonin receptor in the modulation of emotion-based 5. O’mahen HA, Karl A, Moberly N, Fedock G. The association between actions? Mole Neurobiol (2013) 48:841–53. doi: 10.1007/s12035-013-8472-0 childhood maltreatment and emotion regulation: two different mechanisms 14. Meyer JH, Mcmain S, Kennedy SH, Korman L, Brown GM, Dasilva JN, contributing to depression? J Affect Disord (2015) 174:287–95. doi: 10.1016/j. et al. Dysfunctional attitudes and 5-HT2 receptors during depression and jad.2014.11.028 self-harm. Am J Psychiatry (2003) 160:90–9. doi: 10.1176/appi.ajp.160.1.90 6. Padilla Paredes P, Calvete E. Cognitive vulnerabilities as mediators between 15. Stein DJ, Hemmings S, Moolman-Smook H, Audenaert K. 5-HT2A: its role emotional abuse and depressive symptoms. J Abnorm Child Psychol (2014) in frontally mediated executive function and related psychopathology. CNS 42:743–53. doi: 10.1007/s10802-013-9828-7 Spectr (2007) 12:512–6. doi: 10.1017/S1092852900021246 7. Heleniak C, Jenness JL, Stoep AV, Mccauley E, Mclaughlin KA. Childhood 16. Baeken C, Bossuyt A, De Raedt R. Dorsal prefrontal cortical serotonin maltreatment exposure and disruptions in emotion regulation: a 2A receptor binding indices are differentially related to individual scores transdiagnostic pathway to adolescent internalizing and externalizing on harm avoidance. Psychiatry Res Neuroimaging (2014) 221:162–8. doi: psychopathology. Cogn Ther Res (2016) 40:394–415. doi: 10.1007/ 10.1016/j.pscychresns.2013.12.005 s10608-015-9735-z 17. Lopizzo N, Bocchio Chiavetto L, Cattane N, Plazzotta G, Tarazi FI, Pariante 8. Antypa N, Van Der Does AJ. Serotonin transporter gene, childhood CM, et al. Gene–environment interaction in major depression: focus on emotional abuse and cognitive vulnerability to depression. Genes Brain experience-dependent biological systems. Front Psychiatry (2015) 6:68. doi: Behav (2010) 9:615–20. doi: 10.1111/j.1601-183X.2010.00593.x 10.3389/fpsyt.2015.00068

Frontiers in Psychiatry | www.frontiersin.org 11 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

18. Bani-Fatemi A, Howe AS, Matmari M, Koga A, Zai C, Strauss J, et al. abuse and neglect. Am J Psychiatry (1994) 151:1132–6. doi: 10.1176/ Interaction between methylation and CpG single-nucleotide polymorphisms ajp.151.8.1132 in the HTR2A gene: association analysis with suicide attempt in 36. Freeman B, Smith N, Curtis C, Huckett L, Mill J, Craig IW. DNA from buccal schizophrenia. Neuropsychobiology (2016) 73:10–5. doi: 10.1159/000441191 swabs recruited by mail: evaluation of storage effects on long-term stability 19. Abdolmaleky HM, Yaqubi S, Papageorgis P, Lambert AW, Ozturk S, and suitability for multiplex polymerase chain reaction genotyping. Behav Sivaraman V, et al. Epigenetic dysregulation of HTR2A in the brain of Genet (2003) 33:67–72. doi: 10.1023/A:1021055617738 patients with schizophrenia and bipolar disorder. Schizophr Res (2011) 37. Lazary J, Lazary A, Gonda X, Benko A, Molnar E, Juhasz G, et al. New 129:183–90. doi: 10.1016/j.schres.2011.04.007 evidence for the association of the serotonin transporter gene (SLC6A4) 20. Ucok A, Alpsan H, Cakir S, Saruhan-Direskeneli G. Association of a haplotypes, threatening life events, and depressive phenotype. Biol Psychiatry serotonin receptor 2A gene polymorphism with cognitive functions in (2008) 64:498–504. doi: 10.1016/j.biopsych.2008.03.030 patients with schizophrenia. Am J Med Genet B Neuropsychiatr Genet (2007) 38. Team RC. R: A language and environment for statistical computing. Vienna, 144B:704–7. doi: 10.1002/ajmg.b.30463 Austria: R Foundation for Statistical Computing (2013). Available: http:// 21. Lane HY, Liu YC, Huang CL, Hsieh CL, Chang YL, Chang L, et al. Prefrontal www.R-project.org/ [Accessed]. executive function and D1, D3, 5-HT2A and 5-HT6 receptor gene variations 39. Juhasz G, Gonda X, Hullam G, Eszlari N, Kovacs D, Lazary J, et al. Variability in healthy adults. J Psychiatry Neurosci (2008) 33:47–53. in the effect of 5-HTTLPR on depression in a large European population: 22. Saiz PA, Garcia-Portilla MP, Paredes B, Arango C, Morales B, Alvarez V, the role of age, symptom profile, type and intensity of life stressors. PloS One et al. Association between the A-1438G polymorphism of the serotonin 2A (2015) 10:e0116316. doi: 10.1371/journal.pone.0116316 receptor gene and nonimpulsive suicide attempts. Psychiatry Genet (2008) 40. Storey JD, Taylor JE, Siegmund D. Strong control, conservative point 18:213–8. doi: 10.1097/YPG.0b013e3283050ada estimation, and simultaneous conservative consistency of false discovery 23. Banlaki Z, Elek Z, Nanasi T, Szekely A, Nemoda Z, Sasvari-Szekely M, et al. rates: a unified approach.J R Stat Soc Series B (2004) 66:187–205. doi: Polymorphism in the serotonin receptor 2a (HTR2A) gene as possible 10.1111/j.1467-9868.2004.00439.x predisposal factor for aggressive traits. PLoS One (2015) 10:e0117792. doi: 41. Tammen SA, Friso S, Choi SW. Epigenetics: the link between nature and 10.1371/journal.pone.0117792 nurture. Mol Aspects Med (2013) 34:753–64. doi: 10.1016/j.mam.2012.07.018 24. Dong ZQ, Li XR, He L, He G, Yu T, Sun XL. 5-HTR1A and 5-HTR2A genetic 42. Nestler EJ, Pena CJ, Kundakovic M, Mitchell A, Akbarian S. Epigenetic polymorphisms and SSRI antidepressant response in depressive Chinese basis of mental illness. Neuroscientist (2016) 22:447–63. doi: 10.1177/​ patients. Neuropsychiatr Dis Treat (2016) 12:1623–29. doi: 10.2147/NDT. 1073858415608147 S93562 43. Issler O, Chen A. Determining the role of microRNAs in psychiatric 25. Ghadirivasfi M, Nohesara S, Ahmadkhaniha HR, Eskandari MR, Mostafavi disorders. Nat Rev Neurosci (2015) 16:201–12. doi: 10.1038/nrn3879 S, Thiagalingam S, et al. Hypomethylation of the serotonin receptor type-2A 44. Paquette AG, Lesseur C, Armstrong DA, Koestler DC, Appleton AA, Gene (HTR2A) at T102C polymorphic site in DNA derived from the saliva Lester BM, et al. Placental HTR2A methylation is associated with infant of patients with schizophrenia and bipolar disorder. Am J Med Genet B neurobehavioral outcomes. Epigenetics (2013) 8:796–801. doi: 10.4161/ Neuropsychiatr Genet (2011) 156B:536–45. doi: 10.1002/ajmg.b.31192 epi.25358 26. Bonnier B, Gorwood P, Hamon M, Sarfati Y, Boni C, Hardy-Bayle MC. 45. Paquette AG, Marsit CJ. The developmental basis of epigenetic regulation of Association of 5-HT(2A) receptor gene polymorphism with major affective HTR2A and psychiatric outcomes. J Cell Biochem (2014) 115:2065–72. doi: disorders: the case of a subgroup of bipolar disorder with low suicide risk. 10.1002/jcb.24883 Biol Psychiatry (2002) 51:762–5. doi: 10.1016/S0006-3223(01)01228-8 46. Dressler WW, Balieiro MC, Ferreira De Araujo L, Silva WA, Jr., Ernesto Dos 27. Parsons MJ, D’souza UM, Arranz MJ, Kerwin RW, Makoff AJ. The -1438A/G Santos J. Culture as a mediator of gene–environment interaction: cultural polymorphism in the 5-hydroxytryptamine type 2A receptor gene affects consonance, childhood adversity, a 2A serotonin receptor polymorphism, promoter activity. Biol Psychiatry (2004) 56:406–10. doi: 10.1016/j. and depression in urban Brazil. Soc Sci Med (2016) 161:109–17. doi: biopsych.2004.06.020 10.1016/j.socscimed.2016.05.033 28. Myers RL, Airey DC, Manier DH, Shelton RC, Sanders-Bush E. 47. Chang CC, Fang WH, Chang HA, Chang TC, Shyu JF, Huang SY. Polymorphisms in the regulatory region of the human serotonin 5-HT2A Serotonin 2A receptor (5-HT2A) gene promoter variant interacts with receptor gene (HTR2A) influence gene expression. Biol Psychiatry (2007) chronic perceived stress to modulate resting parasympathetic activity 61:167–73. doi: 10.1016/j.biopsych.2005.12.018 in humans. Psychoneuroendocrinology (2017) 76:119–26. doi: 10.1016/j. 29. Gu L, Long J, Yan Y, Chen Q, Pan R, Xie X, et al. HTR2A-1438A/G psyneuen.2016.11.015 polymorphism influences the risk of schizophrenia but not bipolar disorder 48. Woody ML, Mcgeary JE, Gibb BE. Brooding rumination and heart rate or major depressive disorder: a meta-analysis. J Neurosci Res (2013) 91:623– variability in women at high and low risk for depression: group differences 33. doi: 10.1002/jnr.23180 and moderation by COMT genotype. J Abnorm Psychol (2014) 123:61–7. doi: 30. Jin C, Xu W, Yuan J, Wang G, Cheng Z. Meta-analysis of association 10.1037/a0035450 between the -1438a/g (rs6311) polymorphism of the serotonin 2A receptor 49. Mcauley EZ, Fullerton JM, Blair IP, Donald JA, Mitchell PB, Schofield PR. gene and major depressive disorder. Neurol Res (2013) 35:7–14. doi: Association between the serotonin 2A receptor gene and bipolar affective 10.1179/1743132812Y.0000000111 disorder in an Australian cohort. Psychiatr Genet (2009) 19:244–52. doi: 31. Fiocco AJ, Joober R, Poirier J, Lupien S. Polymorphism of the 5-HT2A 10.1097/YPG.0b013e32832ceea9 receptor gene: association with stress-related indices in healthy middle-aged 50. Peterson SM, Thompson JA, Ufkin ML, Sathyanarayana P, Liaw L, Congdon adults. Front Behav Neurosci (2007) 1:3. doi: 10.3389/neuro.08.003.2007 CB. Common features of microRNA target prediction tools. Front Gene 32. Mccaffery JM, Duan QL, Frasure-Smith N, Barhdadi A, Lesperance F, (2014) 5:23. doi: 10.3389/fgene.2014.00023 Theroux P, et al. Genetic predictors of depressive symptoms in cardiac 51. Ding M, Shen W, Hu Y. The Role of miR-539 in the anterior cingulate patients. Am J Med Genet B Neuropsychiatr Genet (2009) 150B:381–8. doi: cortex in chronic neuropathic pain. Pain Med (2017) 18:2433–42. doi: 10.1002/ajmg.b.30824 10.1093/pm/pnx004 33. Derogatis LR. BSI: Brief Symptom Inventory: Administration, Scoring, and 52. Kuhn S, Vanderhasselt MA, De Raedt R, Gallinat J. Why ruminators won’t Procedures Manual. Minneapolis: National Computer Systems Pearson, Inc. stop: the structural and resting state correlates of rumination and its relation to (1993). depression. J Affect Disord (2012) 141:352–60. doi: 10.1016/j.jad.2012.03.024 34. Juhasz G, Dunham JS, Mckie S, Thomas E, Downey D, Chase D, et al. The 53. Baeken C, De Raedt R, Bossuyt A. Is treatment-resistance in unipolar CREB1-BDNF-NTRK2 pathway in depression: multiple gene–cognition– melancholic depression characterized by decreased serotonin2A receptors in environment interactions. Biol Psychiatry (2011) 69:762–71. doi: 10.1016/j. the dorsal prefrontal – anterior cingulate cortex? Neuropharmacology (2012) biopsych.2010.11.019 62:340–6. doi: 10.1016/j.neuropharm.2011.07.043 35. Bernstein DP, Fink L, Handelsman L, Foote J, Lovejoy M, Wenzel K, et al. 54. Walters JT, Owen MJ. Endophenotypes in psychiatric genetics. Mol Initial reliability and validity of a new retrospective measure of child Psychiatry (2007) 12(10):886–90. doi: 10.1038/sj.mp.4002068

Frontiers in Psychiatry | www.frontiersin.org 12 June 2019 | Volume 10 | Article 394 Eszlari et al. HTR2A, Childhood Adversity and Rumination

55. Flint J, Munafo MR. The endophenotype concept in psychiatric genetics. 67. Koren R, Duncan AE, Munn-Chernoff MA, Bucholz KK, Lynskey MT, Psychol Med (2007) 37:163–80. doi: 10.1017/S0033291706008750 Heath AC, et al. Preliminary evidence for the role of HTR2A variants in 56. Bagby RM, Rector NA, Bacchiochi JR, Mcbride C. The stability of the binge eating in young women. Psychiatr Genet (2014) 24:28–33. doi: 10.1097/ response styles questionnaire rumination scale in a sample of patients YPG.0000000000000014 with major depression. Cogn Ther Res (2004) 28:527–38. doi: 10.1023/B:C 68. Watkins ER. Depressive rumination and co-morbidity: evidence for OTR.0000045562.17228.29 brooding as a transdiagnostic process. J Ration Emot Cogn Behav Ther (2009) 57. Gibb BE, Grassia M, Stone LB, Uhrlass DJ, Mcgeary JE. Brooding rumination 27:160–75. doi: 10.1007/s10942-009-0098-9 and risk for depressive disorders in children of depressed mothers. J Abnorm 69. Taylor S. Molecular genetics of obsessive-compulsive disorder: a Child Psychol (2012) 40:317–26. doi: 10.1007/s10802-011-9554-y comprehensive meta-analysis of genetic association studies. Mol Psychiatry 58. Wilkinson PO, Croudace TJ, Goodyer IM. Rumination, anxiety, (2013) 18:799–805. doi: 10.1038/mp.2012.76 depressive symptoms and subsequent depression in adolescents at risk 70. Taylor S. Disorder-specific genetic factors in obsessive-compulsive disorder: for psychopathology: a longitudinal cohort study. BMC Psychiatry (2013) a comprehensive meta-analysis. Am J Med Genet B Neuropsychiatr Genet 13:250. doi: 10.1186/1471-244X-13-250 (2016) 3:325–32. doi: 10.1002/ajmg.b.32407 59. Gottesman Ii, Gould TD. The endophenotype concept in psychiatry: 71. Eszlari N. Biological mechanisms in the background of ruminative response etymology and strategic intentions. Am J Psychiatry (2003) 160:636–45. doi: style. [dissertation]. Budapest, Hungary: Semmelweis University (2018). 10.1176/appi.ajp.160.4.636 72. Eszlari N, Kovacs D, Petschner P, Gonda X, Elliott R, Anderson I, et al. 60. Poddar S, Kesharwani D, Datta M. Interplay between the miRNome and the Brooding subtype of rumination is modulated by the interplay between epigenetic machinery: implications in health and disease. J Cell Physiology serotonin receptor 2A gene and childhood adversity. Eur Neuropsychopharmacol (2017) 232:2938–45. doi: 10.1002/jcp.25819 (2016) 26:S77–S78. doi: 10.1016/S0924-977X(16)70084-8 61. Abernathy DG, Kim WK, Mccoy MJ, Lake AM, Ouwenga R, Lee SW, et al. MicroRNAs induce a permissive chromatin environment that enables Conflict of Interest Statement: RE has received consultancy fees from P1vital and neuronal subtype-specific reprogramming of adult human fibroblasts.Cell Cambridge Cognition. IA has received grant support from AstraZeneca and Servier, Stem Cell (2017) 21:332–48. doi: 10.1016/j.stem.2017.08.002 honorarium for speaking from Lundbeck, and consultancy fees from Alkermes, 62. Swathy B, Banerjee M. Haloperidol induces pharmacoepigenetic response Janssen, Lundbeck/Otsuka, and Servier. JD has performed speaking engagements, by modulating miRNA expression, global DNA methylation and consultancy, and research for P1vital, Autifony, Bristol-Myers Squibb, AstraZeneca, expression profiles of methylation maintenance genes and genes involved Eli Lilly, Janssen-Cilag, Servier, and Schering Plough, with all fees paid to the University in neurotransmission in neuronal cells. PLoS One (2017) 12:e0184209. doi: of Manchester. He also has share options in P1vital. The firms declared above have 10.1371/journal.pone.0184209 not influenced study design, data collection, analysis, interpretation, manuscript 63. Nolen-Hoeksema S, Harrell ZA. Rumination, depression, and alcohol preparation, or decision to submit the paper for publication, in any manner. use: tests of gender differences. J Cogn Psychother (2002) 16:391–403. doi: 10.1891/jcop.16.4.391.52526 The remaining authors declare that the research was conducted in the absence of 64. Caselli G, Ferretti C, Leoni M, Rebecchi D, Rovetto F, Spada MM. Rumination any commercial or financial relationships that could be construed as a potential as a predictor of drinking behaviour in alcohol abusers: a prospective study. conflict of interest. Addiction (2010) 105:1041–8. doi: 10.1111/j.1360-0443.2010.02912.x 65. Cao J, Liu X, Han S, Zhang CK, Liu Z, Li D. Association of the HTR2A gene Copyright © 2019 Eszlari, Petschner, Gonda, Baksa, Elliott, Anderson, Deakin, Bagdy with alcohol and heroin abuse. Hum Genet (2014) 133:357–65. doi: 10.1007/ and Juhasz. This is an open-access article distributed under the terms of theCreative s00439-013-1388-y Commons Attribution License (CC BY). The use, distribution or reproduction in 66. Nolen-Hoeksema S, Stice E, Wade E, Bohon C. Reciprocal relations other forums is permitted, provided the original author(s) and the copyright owner(s) between rumination and bulimic, substance abuse, and depressive are credited and that the original publication in this journal is cited, in accordance symptoms in female adolescents. J Abnorm Psychol (2007) 116:198–207. doi: with accepted academic practice. No use, distribution or reproduction is permitted 10.1037/0021-843X.116.1.198 which does not comply with these terms.

Frontiers in Psychiatry | www.frontiersin.org 13 June 2019 | Volume 10 | Article 394